Attenuated parainfluenza virus (PIV) vaccines

Abstract

The invention provides isolated nucleic acids encoding recombinant genomes or antigenomes of Human Parainfluenza Viruses that are useful as vaccines. The recombinant genomes or antigenomes can be incorporated into expression vectors for production of recombinant viruses in vitro. The invention also provides recombinant Human Parainfluenza viruses having one or more mutations that attenuate replication of the virus in a host.

Description

RELATED APPLICATIONS [0001] This application is a Continuation-In-Part of copending application Ser. No. 09 / 083,793 filed on May 22, 1998, which is a continuation-in-part application of, and claims the benefit under Title 35 of U.S. Provisional Application No. 60 / 047,575, filed May 23, 1997, and U.S. Provisional Application No. 60 / 059,385, filed Sep. 19, 1997. [0002] This application is also a Continuation-In-Part of copending application Ser. No. 09 / 458,813 filed on Dec. 10, 1999, which is a continuation-in-part application of, and claims the benefit under Title 35 of, U.S. patent application Ser. No. 09 / 083,793, filed May 22, 1998 which is a continuation-in-part of U.S. Provisional Application No. 60 / 047,575, filed May 23, 1997, now abandoned, and U.S. Provisional Application No. 60 / 059,385, filed Sep. 19, 1997, now abandoned, and also a Continuation-In-Part of U.S. application Ser. No. 09 / 459,062 filed on Dec. 10, 1999, which is a continuation-in-part application of, and claims t...

AI Technical Summary

Benefits of technology

[0042] Also provided within the invention are recombinant PIV having multiple, phenotype-specifying mutations introduced in selected combinations into the genome or antigenome of an infectious clone to yield desired characteristics including attenuation, temperature sensitivity, cold-adaptation, small plaque size, host range restriction, etc. For example, PIV clones are provided which incorporate at least two separate mutations adopted from a biologically derived PIV mutant, e.g., two ts mutations from HPIV3 JS cp45. Multiply attenuated viruses are thus obtained by selecting mutations from a “menu” of identified lesions and introducing these mutations in various combinations to calibrate a vaccine virus to selected levels of attenuation, immunogenicity and stability.

[0125] Importantly, the presence of multiple PIV serotypes and their unique epidemiology with PIV3 disease occurring at an earlier age than that of PIV1 and PIV2 makes it desirable to sequentially immunize an infant with different PIV vectors each expressing the same heterologous antigenic determinant such as the measles virus HA. This sequential immunization permits the induction of the high titer of antibody to the heterologous protein that is characteristic of the secondary antibody response. In one embodiment, early infants (e.g. 2-4 month old infants) can be immunized with an attenuated chimeric virus of the invention, for example a chimeric HPIV3 expressing the measles virus HA protein and also adapted to elicit an immune response against HPIV3, such as rcp45L(HA P-M). Subsequently, e.g., at four months of age the infant is again immunized but with a different, secondary vector construct, such as the rPIV3-1 cp45L virus expressing the measles virus HA gene and the HPIV1 antigenic determinants as the functional, obligate glycoproteins of the vector. Following the first vaccination, the vaccinee will elicit a primary antibody response to both the PIV3 HN and F proteins and to the measles virus HA protein, but not to the PIV1 HN and F protein. Upon secondary immunization with the rPIV3-1 cp45L expressing the measles virus HA, the vaccinee will be readily infected with the vaccine because of the absence of antibody to the PIV1 HN and F proteins and will develop both a primary antibody response to the PIV1 HN and F protective antigens and a high titered secondary antibody response to the heterologous measles virus HA protein. A similar sequential immunization schedule can be developed where immunity is sequentially elicited against HPIV3 and then HPIV2 by one or more of the chimeric vaccine viruses disclosed herein, simultaneous with stimulation of an initial and then secondary, high titer protective response against measles or another non-PIV pathogen. This sequential immunization strategy, preferably employing different serotypes of PIV as primary and secondary vectors, effectively circumvents immunity that is induced to the primary vector, a factor ultimately limiting the usefulness of vectors with only one serotype. The success of sequential immunization with rPIV3 and rPIV3-1 virus vaccine candidates as described above has been demonstrated. (Tao et al., Vaccine 17:1100-8, 1999).

Problems solved by technology

Infections by these viruses result in substantial morbidity in children less than 3 years of age, and are responsible for approximately 20% of hospitalizations among young infants and children for respiratory tract infections.

Despite considerable efforts to develop effective vaccine therapies against HPIV, no approved vaccine agents have yet been achieved for any HPIV strain, nor for ameliorating HPIV related illnesses.

However, it has not been previously shown which of these identified mutations specify desired, e.g., ts, ca, and att, phenotypes.

Also, this method of complementation does not provide a clear measurement of the relative contribution of the L gene mutation(s) to the overall ts phenotype of cp45.

Despite previous advancements identifying cDNAs for PIV, manipulation of the genomic RNA of this and other negative-sense RNA viruses has proven difficult.

One major obstacle in this regard is that the naked genomic RNA of these viruses is noninfectious.

Rescue of infectious PIV virus and other Mononegavirales members is complicated by virtue of their non-segmented negative-strand RNA genome.

However, PIV and other Mononegavirales members feature much larger and more tightly structured RNPs, which tend to be refractory to functional association in vitro.

Infections by this virus result in substantial morbidity in children less than 3 years of age.

Despite considerable efforts to develop effective vaccine therapies against HPIV, no approved vaccine agents have yet been achieved for any HPIV serotype, nor for ameliorating HPIV related illnesses.

Because these PIV3 candidate vaccine viruses are biologically derived, there is no proven methods for adjusting the level of attenuation should this be found necessary from ongoing clinical trials.

However, to successfully develop vectors for vaccine use, it is insufficient to simply demonstrate a high, stable level of protein expression.

For example, this has been possible since the early-to-mid 1980s with recombinant vaccinia viruses and adenoviruses, and yet these vectors have proven to be disappointments in the development of vaccines for human use.

Similarly, most nonsegmented negative strand viruses which have been developed as vectors do not possess properties or immunization strategies amenable for human use.

Furthermore, some of these prior vector candidates have adverse effects, such as immunosupression, which are directly inconsistent with their use as vectors.

However, the World Health Organization estimates that more than 45 million cases of measles still occur annually, particularly in developing countries, and the virus contributes to approximately one million deaths per year.

Given this need, there have been numerous attempts to develop an immunization strategy to protect infants in the latter half of the first year of life against measles virus, but none of these strategies has been effective to date.

Infection of young infants by aerosol administration of measles virus vaccine was accomplished in highly controlled experimental studies, but it has not been possible to reproducibly deliver a live attenuated measles virus vaccine in field settings by aerosol to the young uncooperative infant (Cutts et al., Biologicals 25, 323-38, 1997).

However, the clinical use of the vaccines in the 1960's revealed a very serious complication, namely, that the inactivated virus vaccines potentiated disease rather than prevented it (Fulginiti et al., JAMA 202:1075-80, 1967).

Initially, this vaccine prevented measles, but after several years vaccinees lost their resistance to infection.

Because of this experience with nonliving measles virus vaccines and also because the immunogenicity of such parenterally-administered vaccines can be decreased by passively-transferred antibodies, there has been considerable reluctance to evaluate such vaccines in human infants.

Replication-competent vaccinia recombinants expressing the protective antigens of RSV have also been shown to be ineffective in inducing a protective immune response when they are administered parenterally in the presence of passive antibody (Murphy et al., J. Virol. 62:3907-10, 1988a), but they readily protected such hosts when administered intranasally.

Unfortunately, replication-competent vaccinia virus recombinants are not sufficiently attenuated for use in immunocompromised hosts such as persons with human immunodeficiency virus (HIV) infection (Fenner et al., World Health Organization, Geneva, 1988; Redfield et al., N. Engl. J. Med. 316, 673-676, 1987), and their administration by the intranasal route even to immunocompetent individuals would be problematic.

Therefore they are not being pursued as vectors for use in human infants, some of whom could be infected with HIV.

Unfortunately, both the immunogenicity and efficacy of MVA expressing a paramyxovirus protective antigen were abrogated in passively-immunized rhesus monkeys whether delivered by a parenteral or a topical route (Durbin et al., Virology 235:323-332, 1999).

In this context, MVA recombinants expressing parainfluenza virus antigens, unlike replication-competent vaccinia virus recombinants, lacked protective efficacy when given by a mucosal route to animals with passively-acquired antibodies, and it is unlikely that they, or the similar avipox vectors, can be used in infants with maternally-acquired measles virus antibodies.

Among the remaining challenges in this context is the need for additional tools to generate suitably attenuated, immunogenic and genetically stable vaccine candidates for use in diverse clinical settings against one or more pathogens.

Images